Cysteine-containing peptide tag for site-specific conjugation of proteins

ABSTRACT

The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety bound to the targeting moiety; the biological conjugate having a covalent bond between the thiol group of SEQ ID NO:2 and a functional group in the binding moiety. The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety that comprises an adapter protein, the adapter protein having a thiol group; the biological conjugate having a disulfide bond between the thiol group of SEQ ID NO:2 and the thiol group of the adapter protein. The present invention is also directed to biological sequences employed in the above biological conjugates, as well as pharmaceutical preparations and methods using the above biological conjugates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part application of U.S. Ser. No.09/872,712 filed Jun. 1, 2001, now abandoned, which claims the benefitof U.S. Provisional Application 60/209,660 filed Jun. 6, 2000, both ofwhich are incorporated by reference herein in their entireties.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made in part with government support under grantnumbers 1 R43 HL61143-01; 1-R43-CA113080-01; R43AI054060-01; and 1 R43GM072170-01 from the National Institutes of Health, and grant numberDE-FG-02-02ER83520 from the Department of Energy. The government hascertain rights in this invention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention is directed to nucleic acid and protein sequencesthat encode a cysteine-containing tag and a targeting protein. Thepresent invention is also directed to a biological conjugate comprisingproteins having the above protein sequences and a binding moietycovalently bound to the cysteine-containing tag in the protein. Thepresent invention is also directed to pharmaceutical compositionscontaining the biological conjugates in combination with selectedtherapeutic, diagnostic, or research entities, and methods ofadministering the pharmaceutical compositions to a patient to achievetargeted delivery of the therapeutic, diagnostic, or research entitiesin the patient. 2. Description of the Related Art.

Conjugation of recombinant proteins to various entities is used inseveral areas. One area is targeted delivery of therapeutic, diagnostic,and research agents to targeted cells in the patient in order to improvetheir efficacy and to minimize potentially adverse side effects. In thisarea either therapeutic, diagnostic, and research agents, or theircarriers are chemically conjugated to recombinant targeting proteinsthat can selectively bind to targeted cells (reviewed by Dubowchik &Walker, 1999). The resulting conjugates are structurally andfunctionally heterogeneous because they are formed randomly via chemicalreactions with few of several available chemical groups, usually ε-aminogroups of lysine residues, in the targeting protein. Since randomconjugation does not discriminate between functionally important anddispensable amino acid residues in the targeting protein, the procedureshould be custom-developed and optimized on a case-by-case basis inorder to increase the proportion of functionally active proteins.

Another area is derivatizing artificial surfaces and/or bulkcompositions of biomedical devices or tissue scaffolds with proteinsthat target certain components of intra-organism environment in order toimprove surface compatibility with the environment and to modulate thedesired features, such as affinity or rejection of certainintra-organism components. In this area recombinant proteins arecovalently grafted on the material through random chemical conjugation,usually via ε-amino groups of lysine residues, that involves bothfunctionally important and dispensable amino acid residues in theproteins, resulting in heterogeneous products with unknown fraction offunctionally active proteins.

Yet another area with similar problems is construction of variousbiosensors or other functional devices with protein-derivatized surfacesthat convert the results of interactions between the “working” proteinand the targeted components of the environment into a detectable output,including but not limited to a detectable signal or the products ofenzymatic activity of immobilized proteins. In this area recombinantproteins are also chemically conjugated to artificial surfaces of thesedevices usually via ε-amino groups of lysine residues, yieldingheterogeneous surfaces with unknown fraction of functionally activeproteins.

Several methods for chemical conjugation of proteins to artificialsurfaces have been developed (see, for example U.S. Pat. No. 5,492,840to Malmqvuist; U.S. Pat. No. 5,853,744 to Mooradian et al.; U.S. Pat.No. 6,033,719 to Keogh, Mann et al. (2001); Kuhl & Griffith-Cima,(1996); Bentz et al. (1998). These methods were developed on acase-by-case basis in order to minimize damage to the protein and toincrease the homogeneity of the surface.

These problems are well recognized, and over the years severalapproaches have been developed for introduction into recombinantproteins of unique cysteine residues for site-specific conjugation ofvarious entities. This strategy is based on observation that intrinsiccysteine residues in proteins are usually involved in intramolecular orinter-subunit disulfide bonds and are not readily available for chemicalconjugation. In theory, introduction of a unique cysteine residue thatdoes not affect formation of intrinsic disulfide bonds and does notaffect functional activity of the recombinant protein can provide athiol group available for site-specific conjugation via chemistriesknown in art. For example, several groups reported introduction ofcysteines into recombinant single-chain Fv antibody fragments (scFv),usually at or near C-termini, in order to use these cysteine residuesfor formation of diabodies and/or for site-specific conjugation tovarious entities (Adams et al., 1993; Kipriyanov et al., 1994; Wang etal., 1997; Marti et al., 2001; Gupta et al., 2001; Xu et al., 2002; Liet al., 2002; Renard et al., 2002; Albrecht et al., 2004). However, evenfor scFv, the presence of unpaired cysteine at or near the C-terminussignificantly affects protein yield, solubility and functional activity(Schmiedl et al., 2000). Futami et al. (2000) introduced cysteineresidues near the N- and C-termini of into human RNase I which resultedin a stabilized RNase I. However, yield and enzymatic activity of theproduct were significantly reduced. Moreover, this mutant RNase I or itsfragments were not used in other products.

Another method for site directed modification of proteins isintein-mediated ligation of various entities to the C-terminus of theprotein (see, for example Evans et al., 1999; Tolbert and Wong 2000;Macmillan et al., 2000; Mukhopadhyay et al., 2001; Hofmann, and Muir,2002; Lovrinovic et al., 2003; Wood et al., 2004). However applicationof this method require proper folding of the protein fused to a largeintein domain and the ability to withstand fairly harsh reducingconditions during intein-mediated ligation. Furthermore, in bothapproaches discussed above, conjugation to available cysteine residue islimited to entities that do not interfere with activities of the proteindespite their close proximity to the body of the protein.

Thus, in the area of protein-based targeted delivery of therapeutic,diagnostic, and research compounds, as well as in the area ofconstruction of various devices and scaffolds with protein-derivatizedsurfaces, what is needed in the art are compositions and general methodsthat (1) allow for site-specific conjugation of recombinant proteins tovarious entities in order to produce more homogeneous products in waysthat minimize interference with functional activities of said proteins;(2) readily convert various recombinant proteins of interest into aformat suitable for site-specific conjugation; (3) can be utilized witha wide variety of entities to which a recombinant protein of interestneed to be conjugated; and (4) do not result in immunogenic or toxicityproblems when introduced into humans. The present invention is believedto be an answer to these objectives.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to an isolatedpolypeptide, consisting of the sequence of SEQ ID NO:2.

In another aspect, the present invention is directed to an isolatednucleic acid consisting of a nucleic acid sequence that encodes thepolypeptide sequence of SEQ ID NO:2.

In another aspect, the present invention is directed to an isolatedpolypeptide, comprising the sequence of SEQ ID NO:4.

In another aspect, the present invention is directed to an isolatednucleic acid comprising a sequence that encodes the polypeptide sequenceof SEQ ID NO:4.

In another aspect, the present invention is directed to an isolatedpolypeptide having an amino acid sequence comprising the polypeptidesequence of SEQ ID NO:2 and the polypeptide sequence of a selectedtargeting protein.

In another aspect, the present invention is directed to an isolatednucleic acid encoding the polypeptide sequence of SEQ ID NO:6, 8, 10,12, or 14.

In another aspect, the present invention is directed to a biologicalconjugate, comprising: (a) a targeting moiety comprising a polypeptidehaving an amino acid sequence comprising the polypeptide sequence of SEQID NO:2 and the polypeptide sequence of a selected targeting protein;and (b) a binding moiety; the biological conjugate having a covalentbond between the thiol group of SEQ ID NO:2 and a functional group inthe binding moiety.

In another aspect, the present invention is directed to a biologicalconjugate, comprising: (a) a targeting moiety comprising a polypeptidehaving an amino acid sequence comprising the polypeptide sequence of SEQID NO:2 and the polypeptide sequence of a selected targeting protein;and (b) a binding moiety that comprises an adapter protein boundcovalently to the targeting moiety, the adapter protein having a thiolgroup; the biological conjugate having a disulfide bond between thethiol group of SEQ ID NO:2 and the thiol group of the adapter protein.

In another aspect, the present invention is directed to a pharmaceucialcomposition for selectively delivering selected entities to a target ina patient, comprising a pharmaceutically acceptable carrier; and one oranother of the above biological conjugates.

In another aspect, the present invention is directed to a method ofselectively delivering entities to a target in a patient, comprising thesteps of: (a) administering to a patient the above pharmaceuticalcomposition; and (b) permitting the biological conjugate to contact thetarget to deliver the entity to the target in the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more fully understood from the following detaileddescription taken in conjunction with the accompanying drawings inwhich:

FIG. 1 is a representation of amino acid and nucleic acid sequences ofC4-tag genetically fused to multiple cloning site region via a G4Slinker (Panel A), and a schematic representation of a plasmid forbacterial expression of fusion recombinant proteins fused to C4-tag viaa G4S linker (Panel B);

FIG. 2 is a schematic representation of site-specific conjugationbetween C4-tagged fusion recombinant protein and a complimentary adapterprotein, named HuS(C118) (Panel A), a schematic representation of afamily of adapter proteins based on human RNase I capable ofsite-specific conjugation to cysteine residue in C4-tag geneticallyfused to recombinant protein (Panel B), a demonstration of spontaneousconjugation via a disulfide bond between C4-VEGF and HuS(C118) leadingto appearance of DTT-sensitive new protein bands in samples namedHuS-C4-VEGF on the SDS-PAGE gel (Panel C), and a demonstration thatribonuclease activity is reconstituted upon chemical conjugateformation, but not upon physical mixing of adapter protein withC4-tagged recombinant fusion protein (Panel D).

FIG. 3 illustrates that functional activities of several C4-taggedrecombinant fusion proteins and C4-tagged recombinant fusion proteinsconjugated to complimentary adapter protein are comparable to that ofparental proteins. Panel A illustrates that in VEGFR-2 tyrosinephosphorylation assay in 293/KDR cells functional activities ofC4-tagged vascular endothelial growth factor, (C4-VEGF), C4-taggedsingle-chain vascular endothelial growth factor (C4-scVEGF), and HuS(C118)-C4-VEGF conjugate (HuS-C4-VEGF) are comparable with that of VEGFthat does not contain C4-tag. Panel B illustrates that functionalactivities of C4-tagged annexin V (C4-annexin) and HUS(C118)-C4-annexinV conjugates (HuS-C4-annexin) are comparable with that of annexin V inan erythrocyte binding assay. Panel C illustrates that ability of aC4-tagged catalytically inactive fragment of anthrax lethal factor,C4-LFn, and the corresponding HuS(C118)-C4-LFn conjugate (HuS-C4-LFn)are comparable to that of LFn in protection of RAW cells from cytotoxicactivity of anthrax toxin LF/PA.

FIG. 4 illustrates site-specific conjugation of HYNIC-maleimide(5-maleimido-2-hydraziniumpyridine hydrochloride), a chelator for^(99m)Tc, to C4-VEGF and C4-scVEGF fusion proteins and functionalactivity of such conjugates. Panel A illustrates that functionalactivities of HYNIC-C4-VEGF and HYNIC-C4-scVEGF conjugates arecomparable to that of parental C4-VEGF in VEGFR-2 tyrosineautophosphorylation assay in 293/KDR cells. Panel B illustrates thatHYNIC-C4-VEGF and HYNIC-C4-scVEGF conjugates are comparable withparental C4-VEGF in their abilities to protect 293/KDR cells fromcytotoxicicity of toxin-VEGF fusion protein.

FIG. 5 illustrates site-specific conjugation of 20 kDa or 40 kDamaleimide-polyethyleneglycol (PEG20, and PEG40, correspondingly) toC4-VEGF and functional activity of the PEG-C4-VEGF conjugate. Panel Aillustrates SDS-PAGE analysis of PEG-C4-VEGF conjugate. Panel Billustrates that functional activity of PEG-C4-VEGF conjugate iscomparable to that of parental C4-VEGF in VEGFR-2 tyrosineautophosphorylation assay in 293/KDR cells.

FIG. 6 illustrates the use of C4-tag and a complimentary adapter proteinHuS(C88, C118) for construction of VEGF-driven conjugates(Cy5.5-Hus-C4-VEGF) containing cyanine dye Cy5.5 for targetednear-infrared fluorescent imaging in vivo. Panel A is a flow-chart forconstruction and characterization of Cy5.5-Hus-C4-VEGF conjugate. PanelB illustrates that functional activity of Cy5.5-Hus-C4-VEGF conjugate iscomparable to that of parental C4-VEGF in VEGFR-2 tyrosineautophosphorylation assay, while site-specific (Cy5.5-C4-VEGF) or randomconjugation (Cy5.5-VEGF) of a similar amount of Cy5.5 per C4-tagged VEGFdecreases activity of VEGF. Panel C illustrates that Cy5.5-Hus-C4-VEGFconjugate is comparable to that of parental C4-VEGF in their ability toprotect 293/KDR cells from cytotoxicity of toxin-VEGF fusion protein,while site-specific (Cy5.5-C4-VEGF) or random conjugation (Cy5.5-VEGF)of a similar amount of Cy5.5 per C4-tagged VEGF decreases VEGF activity.Panel D illustrates the use of Cy5.5-Hus-C4-VEGF conjugate for in vivoimaging of tumor vasculature.

FIG. 7 illustrates the use of C4-tag and a complimentary adapter proteinHuS(C118) for construction of VEGF-driven conjugates (Lip/Hus-C4-VEGF)containing doxorubicin-loaded liposomes (“DOXIL”) for targeted drugdelivery. Panel A is flow-chart for construction and characterization ofLip/Hus-C4-VEGF conjugate. Panel B illustrates that functional activityof Lip/Hus-C4-VEGF conjugate is comparable with that of parental C4-VEGFin VEGFR-2 tyrosine autophosphorylation assay. Panel C illustrates thatVEGF-targeted doxorubicin-loaded liposomes (Lip/HuS-C4-VEGF) are toxicto VEGFR-2 expressing cells in a concentration range where untargeteddoxorubicin-loaded liposomes are ineffective underlyingreceptor-mediated mechanism of toxicity of Lip/Hus-C4-VEGF conjugate.Panel D illustrates that VEGF protects 293/KDR cells from cytotoxicactivity of Lip/Hus-C4-VEGF conjugate.

FIG. 8 illustrates the use of C4-scVEGF for construction of VEGF-drivenconjugates (Lip/C4-scVEGF) containing doxorubicin-loaded liposomes(“DOXIL”) for targeted drug delivery. Panel A is a flow-chart forconstruction and characterization of Lip/C4-scVEGF conjugate. Panel Billustrates that in VEGFR-2 tyrosine autophosphorylation assayfunctional activity of Lip/C4-scVEGF conjugate is comparable with thatof parental C4-VEGF. Panel C illustrates that Lip/C4-scVEGF are toxic toVEGFR-2 expressing cells in a concentration range where untargeteddoxorubicin-loaded liposomes are ineffective underlyingreceptor-mediated mechanism of toxicity of Lip/C4-scVEGF conjugate.Panel D illustrates that VEGF protects 293/KDR cells from cytotoxicactivity of Lip/C4-scVEGF conjugate.

DETAILED DESCRIPTION OF THE INVENTION

As indicated above, the present invention comprises compositions andmethods useful for site-specific conjugation of recombinant fusionproteins to various entities via a single cysteine residue present in apeptide tag engineered into the protein. Current conjugation methodsknown in the art rely mostly on random cross-linking of various entitiesto amino acid residues, such as, for example, lysine or tyrosine, thatare abundant in the protein. Less abundant cysteine residues are usuallyinvolved in intramolecular disulfide bonds essential for the functionalactivity of the protein and therefore not available for conjugation. Asa result, even when conjugation involves only one amino acid residue perprotein, the final product contains a mixture of proteins modified atdifferent positions and therefore heterogeneous in their activity,pharmacokinetics, pharmacodynamic, and tissue distributioncharacteristics. Furthermore, conjugation to amino acid residues in theprotein is always limited by the harm it may inflict upon the functionalactivity of the proteins. As a result, conjugation procedures have to becustom-developed on a case-by-case basis. However, customizedconjugation does not allow a standardized approach to rapid adaptationof different proteins for similar purposes, for example for a targeteddelivery of the same imaging reagent, or surface derivatization of thesame device.

To overcome these and other obstacles, the present invention disclosescompositions and methods useful for site-specific conjugation ofrecombinant proteins to various entities. The method is based onconversion of a protein of interest into a fusion protein that comprisesa cysteine-containing peptide tag, named C4, fused via a linker peptideto an N-terminus or a C-terminus of the protein of interest. Bydefinition, in order to be useful, such fusion proteins should retainits functional activities, and a cysteine residue in the peptide tagshould be available for site-specific conjugation with various entities,including complimentary adapter proteins, capable of forming a covalentdisulfide bond with tag's cysteine. Various chemistries for conjugationto a cysteine thiol group are well-known in art. For example, entitiesderivatized with maleimide groups, or vinylsulfone groups, or withchemically activated thiol groups can be conjugated to a thiol group inC4-tag. On the other hand, thiol group in the C4-tag can be modifiedwith formation of an activated disulfide bond that might react withavailable thiol groups on various entities, or it can be modified withbifunctional reagents for conjugation to entities that can react withthe second functional group.

Thus, it has now been discovered that a cysteine-containing peptidefusion C4-tag can confer upon a protein fused to the tag the ability tobe chemically conjugated to various chemical components. The cysteineresidue of the C4 tag is used to form a covalent bond to the chemicalcomponents, thus providing a strong, stable linkage. Through thecysteine residue of C4, stable bonds may be formed between the C4 taggedprotein and a wide variety of entities, including drugs, drug carriers,contrast agents, carriers for contrast agents, radionuclides, carriersfor radionuclides, various nano- and microparticles, includingliposomes, quantum dots, small and ultra-small paramagnetic particles,other proteins or protein fragments, nucleic acids, variousprotein-modifying molecules, including but not limited topolyethyleneglycol of various molecular weights, as well as surfaces ofvarious devices, such as biomedical devices, biosensors, or artificialtissue scaffolds. In one embodiment, a complimentary adapter protein iscovalently bonded to the C4 tagged protein through a disulfide bond. Theadapter protein can be used a platform for conjugation to variousentities described above. The present invention discloses that C4-taggedproteins retain protein functional activity and can be site-specificallyconjugated to various entities without loss of functional activityeither in vitro or in vivo.

The formation of a covalent bond offers several advantages for thepresent invention. First, the entity covalently bound to C4-tag isremoved from the functional parts of the protein. Furthermore, acomplimentary adapter protein allows further distancing of boundentities from the functional parts of the protein. Second, the covalentbond is strong such that the materials bound to the C4 tagged protein donot easily dissociate in biological fluids or in solutions. Third, thechemistry and conditions to form the covalent bond known and can beeasily reproduced. Fourth, under certain conditions known to those ofskill in the art, it is possible to destroy the covalent linkage andpermit the components to dissociate. For example, if a disulfide bond isformed, appropriate conditions can be selected to reduce this bond andpermit the C4-tagged protein to dissociate. This feature of theinvention offers advantages in that, for example, that derivatizedsurfaces can be regenerated after a predetermined length of time.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in artto which the subject matter herein belongs.

As used herein, “C4”, refers to a mutant N-terminal 15 amino acid longfragment of human RNase I with the following nucleic acid and amino acidsequences:

(SEQ ID NO: 1) AAAGAATCTTGCGCTAAAAAATTTCAACGTCAACACATGGACTCT (SEQ ID NO:2) Lys-Glu-Ser-Cys-Ala-Lys-Lys-Phe-Gln-Arg-Gln-His- Met-Asp-Ser

Although minimally altered human peptides are not expected to beimmunogenic in humans and therefore are preferred embodiments ofcysteine-containing tags, it is understood that in some applicationsimmunogenicity is not an issue, and therefore these peptides may be usedas modified by amino acid substitutions, amino acid deletions, aminoacid insertions, and amino acid additions, including placing of cysteineresidue in different positions in these peptides or using correspondingfragments from non-human ribonucleases.

As used herein, “targeting protein” refers to a protein that canselectively interact with cellular receptors or other cell surfaceproteins, or can selectively interact with certain components of theenvironment, either free or bound to a surface.

As used herein, the term “linker sequence” refers to an innocuous lengthof nucleic acid or protein that joins two other sections of nucleic acidor protein.

The terms “mutant” and “mutated” refer to nucleic acid or proteinsequences which are not found in nature. The term “truncated” refers tonucleic acid or protein sequences that are shorter than those found innature.

As defined herein, “biological conjugate” refers to a complex between atargeting moiety and a binding moiety and stabilized by a covalent bond.“Targeting moiety” refers to a protein having a polypeptide comprisingC4 and a targeting protein. “Binding moiety” refers to any substance orsurface that can be covalently bound to the targeting moiety. The term“functional group” refers to natural or non-natural chemical groups thatinteract chemically with a thiol group. A natural functional grouprefers to a chemical group that is found naturally in the binding moietythat can interact chemically with a thiol group. A non-naturalfunctional group refers to a group that is artificially added to thebinding moiety (e.g., addition of a maleimide group or vinylsulfonegroup to polyethylene glycol) such that the artificial group ischemically reactive with the thiol group of the binding moiety.

“Adapter protein” refers to a protein that is complimentary (e.g., partof a binding pair) and can interact with the targeting moiety to form aspecific disulfide bond between it and the targeting moiety. “Fusionprotein” refers to a recombinant protein that contains two or morepolypeptide fragments that are encoded by DNA sequences that have beencombined with the methods of recombinant DNA technology in a form thatallows expression of the fusion protein in suitable hosts.

As used herein, “carrier” refers to natural or synthetic molecules oraggregates thereof which can be associated covalently or non-covalentlywith therapeutic, diagnostic, or research compounds. Such carriersinclude, but are not limited to chelators, natural or synthetic polymersincluding dendrimers, co-polymers, derivatized polymers, liposomes,various viral and bacteriophage particles, various natural andmanufactured nano- and microparticles, and beads.

As used herein, “scVEGF” refers to a single chain vascular endothelialgrowth factor that comprises two 3 to 112 amino acid residue fragmentsof the VEGF₁₂₁ isoform connected head-to-tail into a single-chainprotein via alanine residue and has the following protein and nucleicacid sequences:

(SEQ ID NO:4)    NH₂-Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val ValLys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu ValAsp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser CysVal Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys ValPro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His GlnGly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu Cys ArgPro Lys Lys Asp Arg Ala Arg Ala Met Ala Glu Gly Gly Gly Gln Asn His HisGlu Val Val Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile GluThr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe LysPro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly LeuGlu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile LysPro His Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys CysGlu Cys Arg Pro Lys Lys Asp Arg Aia Arg- COOH (SEQ ID NO:3)     ATGGCAGAAGGAGGAGGGCAGAATCATCACGAAGTGGTGAAGTTCATGGATGTCTATCAGCGCAGCTACTGCCATCCAATCGAGACCCTGGTGGACATCTTCCAGGAGTACCCTGATGAGATCGAGTACATCTTCAAGCCATCCTGTGTGCCCCTGATGCGATGCGGGGGCTGCTGCAATGACGAGGGCCTGGAGTGTGTGCCCACTGAGGAGTCCAACATCACCATGCAGATTATGCGGATCAAACCTCACCAAGGCCAGCACATAGGAGAGATGAGCTTCCTACAGCACAACAAATGTGAATGCAGACCAAAGAAAGATAGAGCAAGAGCCATGGCAGAAGGAGGAGGGCAGAATCATCACGAAGTGGTGAAGTTCATGGATGTCTATCAGCGCAGCTACTGCCATCCAATCGAGACCCTGGTGGACATCTTCCAGGAGTACCCTGATGAGATCGAGTACATCTTCAAGCCATCCTGTGTGCCCCTGATGCGATGCGGGGGCTGCTGCAATGACGAGGGCCTGGAGTGTGTGCCCACTGAGGAGTCCAACATCACCATGCAGATTATGCGGATCAAACCTCACCAAGGCCAGCACATAGGAGAGATGAGCTTCCTACAGCACAACAAATGTGAATGCAGACCAAAGAAAGATAGAGCAAGA TGA

Due to the redundancy of the genetic code, however, any nucleic acidsthat code for SEQ ID NO:4 is embraced by the nucleic acids of thepresent invention.

In yet another embodiment, the targeting moiety has the polypeptidesequence shown in SEQ ID NO: 14. This polypeptide sequence, which is agenetic fusion of C4 and a single chain VEGF (scVEGF) having thepolypeptide sequence shown in SEQ ID NO:4, is encoded by the nucleicacid sequence shown in SEQ ID NO:13. Due to the redundancy of thegenetic code, however, any nucleic acids that code for SEQ ID NO:14 isembraced by the nucleic acids of the present invention. Although scVEGFcontains 16 cysteine residues per single-chain molecule, C4-taggedmolecules are refolded into functionally active conformation, wherebythe cysteine residue in C4-tag can be conjugated to various entitiesyielding conjugates with functional activities comparable to that ofunmodified VEGF.

As indicated above, in one embodiment, the present invention is directedto a biological conjugate, comprising: (a) a targeting moiety comprisinga polypeptide having an amino acid sequence comprising the polypeptidesequence of SEQ ID NO:2 and the polypeptide sequence of a selectedtargeting protein; and (b) a binding moiety; wherein the biologicalconjugate has a covalent bond between the thiol group of SEQ ID NO:2 anda functional group in the binding moiety. The present invention is alsodirected to a biological conjugate, comprising: (a) a targeting moietycomprising a polypeptide having an amino acid sequence comprising thepolypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of aselected targeting protein; and (b) a binding moiety that comprises anadapter protein bound covalently to the targeting moiety, the adapterprotein having a thiol group; wherein the biological conjugate has adisulfide bond between the thiol group of SEQ ID NO:2 and the thiolgroup of the adapter protein. Each of these components are discussed inmore detail below.

The targeting moiety is preferably a protein having a polypeptidecomprising the C4 peptide and a targeting protein. As indicated above,the C4 peptide portion of the targeting moiety is a mutant N-terminal 15amino acid long fragment of human RNase I wherein arginine at position 4has been substituted with cysteine. This particular peptide offersseveral advantages in the present invention. The human origin decreasesthe likelihood of inducing a strong immune response in a human host.Furthermore, an N-terminal fragment of human RNase I is capable offorming an α-helix that may protect it from forming disulfide bonds withother cysteine residues in the fusion protein during refolding andpurification of the protein. Finally, enzymatically inactive wild typeN-terminal and C-terminal fragments of human RNase I spontaneously formenzymatically active non-covalent complexes, a phenomenon that isexploited in the present invention for developing of complimentaryadapter proteins, capable of forming disulfide bond with C4 residue inC4-tag. The mutant N-terminal 15 amino acid long fragment of human RNaseI has the following nucleic acid and amino acid sequences:

(SEQ ID NO:1) AAAGAATCTTGCGCTAAAAAATTTCAACGTCAACACATGGACTCT (SEQ IDNO:2) Lys-Glu-Ser-Cys-Ala-Lys-Lys-Phe-Gln-Arg-Gln-His- Met-Asp-Ser

The targeting protein portion of the targeting moiety is any proteinthat can selectively bind to cellular receptors or other cell surfaceproteins or selectively interact with certain components of theenvironment, and is genetically fused to the C4 peptide. In preferredembodiments, the targeting protein may be human vascular endothelialgrowth factor (VEGF), or a mutated or truncated form thereof such asVEGF₁₁₀, human annexin V, or a mutated or truncated form thereof, acatalytically inactive fragment of anthrax lethal vector, or a mutatedor truncated form thereof, or a single chain VEGF derivative, or amutated or truncated form thereof.

In one embodiment, the targeting moiety has the polypeptide sequencesshown in SEQ ID NOS: 6 or 8. These polypeptide sequences, which aregenetic fusions of C4 and VEGF₁₂₁ or VEGF₁₁₀, respectively, are coded bythe nucleic acid sequences shown in SEQ ID NOS:5 and 7, respectively.Due to the redundancy of the genetic code, however, any nucleic acidsthat code for SEQ ID NOS:6 or 8 are embraced by the nucleic acids of thepresent invention. In addition, although selected isoforms of VEGF(VEGF₁₂₁) contains 18 cysteine residues per dimeric molecule, taggedmolecules are refolded into functionally active conformation, wherebycysteine residue in C4-tag can be conjugated to various entitiesyielding conjugates with functional activities comparable to that ofunmodified VEGF.

Vascular endothelial growth factor (VEGF) controls growth of endothelialcells via interaction with several receptors, among which KDR/flk-1(VEGFR-2) receptor expression is limited mostly to endothelial cells. Inadult organisms the growth of endothelial cells (angiogenesis) occurs,with the exception of corpus luteum development, only in variouspathological conditions. Thus, KDR/flk-1 (VEGFR-2) receptor-mediateddelivery of therapeutic, diagnostic, contrast, and research entitiessite-specifically linked to VEGF or scVEGF, or linked to VEGF or scVEGFvia a complimentary adapter protein, may be useful in therapies forvarious pathologies. On the other hand, long-circulating, orslow-releasable from a suitable matrix, site-specifically PEGylated VEGFor scVEGF, as well as VEGF or scVEGF conjugated to the surfaces ofbiomedical devices, such as stents, or tissue scaffolds, might be usefulfor promotion of angiogenesis in ischemic situations.

In another embodiment, the targeting moiety has the polypeptide sequenceshown in SEQ ID NO:10. This polypeptide sequence, which is a geneticfusion of C4 and annexin V, is encoded by the nucleic acid sequenceshown in SEQ ID NO:9. Due to the redundancy of the genetic code,however, any nucleic acids that code for SEQ ID NO:10 is embraced by thenucleic acids of the present invention. Although annexin V contains asingle cysteine residue, tagged molecules are refolded into functionallyactive conformation, whereby the cysteine residue in C4-tag can beconjugated to various entities yielding conjugates with functionalactivities comparable to that of unmodified annexin V. Annexin Vinteracts with phosphatidylserine exposed on the surface of apoptoticcells, and is used as an early marker of apoptotic process. Thus,phosphatidylserine-mediated delivery of therapeutic, diagnostic, orresearch entities site-specifically linked to annexin V might be usefulfor inhibition or promotion of apoptosis.

In another embodiment, the targeting moiety has the polypeptide sequenceshown in SEQ ID NO:12. This polypeptide sequence, which is a geneticfusion of C4 and a catalytically inactive fragment of anthrax lethalfactor, known as LFn, is encoded by the nucleic acid sequence shown inSEQ ID NO:11. Due to the redundancy of the genetic code, however, anynucleic acids that code for SEQ ID NO:12 is embraced by the nucleicacids of the present invention. Although LFn contains no cysteineresidues, tagged molecules are refolded into functionally activeconformation, whereby the cysteine residue in C4-tag can be conjugatedto various entities yielding conjugates with functional activitiescomparable to that of unmodified LFn. Catalytically inactive fragment ofanthrax lethal factor, LFn, pairs with another anthrax protein, namedprotective antigen (PA) that interacts with the same cellular receptorsas the combination of catalytically active lethal factor/protectiveantigen (LF/PA). Thus, PA-mediated delivery of therapeutic, diagnostic,or research entities site-specifically linked to LFn might be useful formapping sites with receptors for PA, or delivery to cells compounds thatmight interfere with cytotoxic activity of lethal factor.

In yet another embodiment, the targeting moiety has the polypeptidesequence shown in SEQ ID NO:14. This polypeptide sequence, which is agenetic fusion of C4 and a single chain VEGF (scVEGF) having thepolypeptide sequence shown in SEQ ID NO:4, is encoded by the nucleicacid sequence shown in SEQ ID NO:13. Due to the redundancy of thegenetic code, however, any nucleic acids that code for SEQ ID NO:14 isembraced by the nucleic acids of the present invention. As indicatedabove, scVEGF comprises two 3 to 112 amino acid residue fragments of theVEGF₁₂₁ isoform connected head-to-tail into a single-chain protein viaalanine residue. Although scVEGF contains 16 cysteine residues persingle-chain molecule, tagged molecules are refolded into functionallyactive conformation, whereby the cysteine residue in C4-tag can beconjugated to various entities yielding conjugates with functionalactivities comparable to that of unmodified dimeric VEGF.

With reference to the above targeting moiety, a linker sequence may bepositioned between the C4 peptide and the sequence of the targetingprotein. Linker sequences, such as Gly₄Ser or (Gly₄Ser)₃ linkers, areengineered in the commercially available vectors for bacterialexpression of recombinant proteins and can be readily engineered intovectors for expression of recombinant proteins in other hosts,including, but not limited to mammalian cells, insect cells, yeastcells, and transgenic organisms. Linkers serve to provide some usefuldistance between the C4 peptide and the targeting protein. Although inthe presented embodiments C4-tag is positioned at the N-terminus oftargeting protein, one skilled in the art would appreciate that the tagmay be placed at the C-terminus of the targeting protein, or inside thefunctionally dispensable area of targeting protein, for example betweentwo functional domains of single-chain antibody, using commonly knownmethods of genetic engineering.

FIG. 1, Panel A, shows amino acid and nucleic acid sequences of C4-taggenetically fused to a multiple cloning site region found in the typicalexpression plasmid via a G4S linker. As shown in Panel A of FIG. 1, thefull nucleic acid sequence includes control and transcription elements(T7 promotor, lac operatior, ribosome binding site, and the like) aswell as the nucleic acid sequence of the C4 tag. A linker sequenceseparates the C4 sequence from a multiple cloning site, which may beused to introduce nucleic acids of interest that will function as thetargeting protein. A T7 termination site completes the full lengthnucleic acid. FIG. 1, Panel B, shows a schematic representation of aplasmid for bacterial expression of fusion recombinant proteins fused toC4-tag via a G4S linker.

The targeting moiety of biological conjugate may be cytokines,chemokines, growth factors, antibodies and their fragments, enzymes, andcombinations of thereof that may be useful in various biomedical orindustrial applications. The binding moiety portion of the biologicalconjugate may be any substance or surface that can be covalently boundto the C4-thiol group of targeting moiety or to functional group ofadapter protein in the adapter/targeting moiety conjugate. Examples ofuseful binding moieties include, but are not limited to, drugs,radionuclide chelators, polyethylene glycol, dyes, lipids, liposomes,and selected surfaces.

Useful radionuclide chelators include compounds such as5-maleimido-2-hydraziniumpyridine hydrochloride (HYNIC) for loading withimaging or therapeutic radionuclide. Polyethylene glycol is useful forslowing blood clearance of protein, and may be used in a derivatized(e.g., modified with maleimide or vinylsulfone) or underivatized forms.Useful dyes include, for example, cyanine dye Cy5.5 for near-infraredfluorescent imaging. Surfaces that may bind to the C4-thiol group oftargeting moiety or to functional group of adapter protein in theadapter/targeting moiety conjugate include surfaces of a nano- ormicroparticle, the surface of a dendrimer, surfaces of tissue culturescaffolds, biomedical devices, and biosensors. It is understood thatwhen a binding entity include surface, it may be used as such, or mayhave other chemical groups deposited on the surface by methods known inart. These chemical groups might be further used for modification withbifunctional reagents that allow conjugation to C4-tag or to adapterprotein via methods known in art.

In one embodiment of the present invention, the biological conjugate hasa covalent bond between the thiol group of the C4 peptide (SEQ ID NO:2),and a naturally occurring functional group in the binding moiety. Thus,it is contemplated that the thiol group of the C4 peptide reactschemically directly with a reactive group in the binding moiety suchthat a stable covalent bond is formed. In alternative embodiments, thereactive group that reacts with the thiol group of the C4 peptide may beintroduced artificially, for example by using bifunctional crosslinkingagents known in art. For example, a maleimide group can be introducedinto polyethylene glycol or a lipid and used for reaction with the thiolgroup of the C4 peptide.

In an alternative embodiment, the biological conjugate of the presentinvention includes a binding moiety that comprises adapter protein boundcovalently to the targeting moiety. In general, the adapter protein is amutant human RNase I, or a fragment thereof, that includes cysteine atposition 118 (Cys₁₁₈). Examples of adapter proteins useful in thepresent invention include proteins having polypeptide sequences shown inSEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22, which aredescribed in more detail below.

Four particularly preferred embodiments of adapter protein capable offorming a disulfide bond with C4 residue in C4-tag are shown in FIG. 2where Panel A provides a schematic representation of site-specificconjugation between C4-tagged fusion recombinant protein and acomplimentary adapter protein. FIG. 2, Panel B shows a schematicrepresentation of a family of adapter proteins based on T24N, V118Cmutant human RNase I. The first embodiment is a fragment of T24N, V118Cmutant human RNase I, named HuS(C118) (SEQ ID NOS:15 and 16). Aminogroups of HuS(C118) can be used for conjugation to various entitiesusing methods known in art prior to site-specific conjugation ofHuS(C118) to C4-tagged proteins. The second embodiment is a fragment ofT24N, V118C mutant human RNase I containing the N88C substitution, namedHuS(C88, C118) (SEQ ID NOS:17 and 18). C88 thiol group in HuS(C88, C118)can be used for conjugation to various entities using methods known inart after site-specific conjugation of HuS(C88, C118) to C4-taggedproteins. The third and fourth embodiments are based on a chimericBH-RNase that contains a 1-29-aa fragment of bovine RNase A that differsfrom a corresponding fragment of human Rnase I in several positions anda 30-127 aa fragment of human RNase I (Gaynutdinov et al., 2003). Thethird embodiment is the V118C mutant of BH-RNase containing the F8A andQ11P, substitutions, named BHR(A8, P11, C118) (SEQ ID NOS:19 and 20)which does not require removal of 20 N-terminal amino acid residuesprior to site-specific conjugation to C4-tagged proteins. Amino groupsof BHR(A8, P11, C118) can be derivatized with various entities usingmethods known in art prior to site-specific conjugation of HuR(A8, P11,C118) to C4-tagged proteins. The fourth embodiment is the V118C mutantBH-RNase containing A8, P11, and C88, named BHR(A8, P11,C88, C118) (SEQID NOS:21 and 22) which does not require removal of 20 N-terminal aminoacid residues prior to site-specific conjugation to C4-tagged proteins.The C88 thiol group in BHR(A8, P11,C88, C118) can be used forconjugation with various entities using methods known in art aftersite-specific conjugation of BHR(A8, P11,C88, C118) to C4-taggedproteins. Using methods known in art it will be easy to construct otheradapter proteins based on human RNase I capable of site-specificconjugation to C4-tagged proteins and providing convenient platforms forderivatization with various entities. One of skill in the art wouldappreciate that other adapter proteins based on human RNase I may beused in addition to those explicitly described herein. For example, oneof skill in the art would appreciate that adapter proteins based onother ribonucleases, for example bovine RNase A, may be constructed aslong as these adapter proteins retain the ability to form conjugateswith C4-tag, or similar tags based on N-terminal fragments of otherribonucleases.

Panel C in FIG. 2 provides evidence of C4-VEGF and HuS(C118) conjugationvia a disulfide bond leading to the appearance of DTT-sensitive newprotein bands in samples named HuS-C4-VEGF on the SDS-PAGE gelcorresponding to conjugation of one or two adapter HuS(C118) to C4-tagsin a dimeric molecule of C4-VEGF. Panel D provides evidence thatribonuclease activity is reconstituted upon chemical conjugateformation, but not upon physical mixing of HuS(C118) adapter proteinwith C4-tagged recombinant fusion protein (Panel D).

The adapter protein may be derivatized with a host of functionalmaterials to achieve a desired result. For example, in one embodiment,the adapter protein may be derivatized with a dye, such as Cy5.5 dye foroptical imaging, or with a lipid for associating the adapter proteinwith a liposome that acts as a carrier for therapeutic, diagnostic, orresearch compounds. In one embodiment, the liposomes may carry or beloaded with doxorubicin (“DOXIL”).

The biological conjugates may be combined with pharmaceuticallyacceptable carriers to produce pharmaceutical compositions forselectively delivering therapeutic, research, or diagnostic compounds toa target in a patient. Such a pharmaceutical composition may beadministered to a patient, and the biological conjugate is permitted tocontact the target to deliver the compound to the target in the patient.In these embodiments, useful pharmaceutically acceptable carriersinclude materials such as water, gelatin, lactose, starch, magnesiumstearate, talc, plant oils, gums, alcohol, Vaseline, or the like. Thepharmaceutical preparation of the invention should include an amount ofthe biological conjugate effective for the desired activity. Theeffective dosage will depend on the activity of the particularbiological conjugate employed and is thus within the ordinary skill ofthe art to determine for any particular host mammal or other hostorganism.

In general, a pharmaceutically effective amount of the biologicalconjugate is combined in a conventional fashion with thepharamaceutically acceptable carrier to produce the pharmaceuticalcomposition. The pharmaceutical composition of the invention ispreferably administered internally, e.g., intravenously, in the form ofconventional pharmaceutical preparations, for example in conventionalenteral or parenteral pharmaceutically acceptable excipients containingorganic and/or inorganic inert carriers as described above. Thepharmaceutical preparations can be in conventional solid forms, forexample, tablets, dragees, suppositories, capsules, or the like, orconventional liquid forms, such as suspensions, emulsions, or the like.If desired, they can be sterilized and/or contain conventionalpharmaceutical adjuvants, such as preservatives, stabilizing agents,wetting agents, emulsifying agents, buffers, or salts used for theadjustment of osmotic pressure. The pharmaceutical preparations may alsocontain other therapeutically active materials.

EXAMPLES

The following Examples are intended to illustrate, but in no way limitthe scope of the present invention. All parts and percentages are byweight and all temperatures are in degrees Celsius unless explicitlystated otherwise.

Example 1 Construction and use of Expression Vectors Containing C4-and aLinker Peptide

1. Bacterial Strains and Plasmids

E. coli strain DH5α-T1 is commercially available from Invitrogen (USA).E. coli strains B121(DE3)and NovaBlue are commercially available fromNovagen (USA). The pET29a(+) bacterial expression vector was obtainedfrom Novagen (USA). The pLen-121 plasmid containing the DNA sequenceencoding the 121-amino acid residue isoform of human VEGF has beendescribed in U.S. Pat. No. 5,219,739, herein incorporated by referencein its entirety, and was obtained from Dr. J. Abraham (Scios Nova, Inc.,USA). The pPAP1-1.6 plasmid containing the DNA sequence encoding humanannexin V was obtained from Dr. J. Tait (University of Washington Schoolof Medicine, Seattle, Wash.). The pGEX-KG LF254 plasmid containing theDNA sequence encoding non-toxic N-terminal fragment of anthrax lethalfactor (LFn) was obtained from Dr. S. Leppla (National Institute ofAllergy and Infectious Diseases, NIH, Bethesda, Md.).

2. DNA Manipulations

The restriction and modification enzymes employed herein arecommercially available in the U.S. and were used according to themanufacturer's instructions. Preparation of competent cells,transformation, and bacterial medium were according to Sambrook et al.(J. Sambrook, E. F. Fritsch and T. Maniatis. (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.) or according to the manufacturer's instructions.Purification of plasmids was done using Wizard Plus SV Minipreps orMaxipreps DNA Purification Systems (Promega, USA) according to themanufacturer's instructions. The purification of DNA fragments fromagarose gels was done using the Geneclean Spin kit (Bio 101, USA)according to the manufacturer's instructions.

3. Construction of Vectors for Expression of Proteins Fused to C4-Tag.

The pET/Hu(G4S)3 vector for expression of recombinant proteins with anN-terminal Hu-tag fused to protein ORF via a (G₄S)₃ linker wasconstructed as described (Backer et al., 2004). The R4C amino acidsubstitution was introduced in Hu-tag of the pET/Hu(G4S)₃ vector bysite-directed mutagenesis using Gene-Tailor Site Directed mutagenesiskit (Invitrogen). Primers (SEQ ID NO:23 and SEQ ID NO:24) were used forintroducing the R4C mutation. The resulted vector was confirmed bysequencing and designated pET/Hu-C4(G4S)₃.

SEQ ID NO:23 5′-ATATACATATGAAAGAATCTTGCGCTAAAAAATTTC SEQ ID NO:245′-AGATTCTTTCATATGTATATCTCCTTCTTAAAGTT

DNA encoding C4-G₄S was amplified by PCR using pET/Hu-C4(G₄S)₃ vectorDNA as a template, a sense primer SEQ ID NO:25 introducing XbaI site,and antisense primer SEQ ID NO:26 introducing NcoI site. The insert wascloned into the pET29a(+) vector cut with XbaI and NcoI. The resultingvector was confirmed by sequencing and designated pET/Hu-C4-G₄S.

SEQ ID NO:25 5′-GGATAACAATTCCCCTCTAGAAATAATTTTGTTTAAC SEQ ID NO:265′-ACTACCCATGGAACCGCCGCCACCAGAGTCCATG

4. Sub-Cloning of cDNA Fragments Encoding Proteins of Interest into thepET/Hu-C4(G₄S)₃ and pET/Hu-C4-G₄S Vectors.

The pET/Hu-C4(G₄S)₃ and pET/Hu-C4-G₄S vector DNAs were cut separatelywith NcoI and dephosphorylated. All DNA fragments to be cloned in theabove vectors were amplified by PCR using primers introducing NcoI sitesto the both ends of the amplified DNA fragments. DNA encoding the 121-aaisoform of human VEGF was amplified from the pLen-121 plasmid DNA usingprimers (SEQ ID NO:27 and SEQ ID NO:28). DNA encoding a 1-254-aa longN-terminal fragment of anthrax lethal factor (LFn) was amplified fromthe pGEX-KG LF254 plasmid DNA using primers (SEQ ID NO:29 and SEQ IDNO:30). DNA encoding human annexin V was amplified from the pPAP1-1.6plasmid using primers (SEQ ID NO:31 and SEQ ID NO:32). Each amplifiedDNA fragment was cut with NcoI, purified, and cloned into NcoI sites ofthe vectors pET/Hu-C4(G₄S)₃ and pET/Hu-C4-G₄S. Clones with correctorientations of cloned ORF were selected by PCR using a T7-promoterbased sense primer, and confirmed by sequencing.

SEQ ID NO:27 5′-CACAAGCCATGGCACCCATGGCAGAAGGAGGA SEQ ID NO:285′-ACTACCCATGGTCACCGCCTCGGCTTGTCAC SEQ ID NO:295′-CTGCTCCATGGGAGCGGGCGGTCATGGTGATG SEQ ID NO:305′-ACTACCCATGGCTATAGATTTATTTCTTGTTCGTTAAATTTATC SEQ ID NO:315′-CACAAGCCATGGCACAGGTTCTCAGAG SEQ ID NO:325′-ACTACCCATGGTTAGTCATCTTCTCCACAGAGC

5. Expression and Purification of Recombinant Fusion Proteins withC4-Tag.

All proteins were expressed in BL21(DE3) E. coli grown in LB medium(Q-Biogen, Carlsbad, Calif.). The expression was induced by 1 mM IPTG atthe optical density of 0.5-0.7 unit at 600 nm. After induction, cultureswere grown for 2.5-3 hrs at 37° C. with shaking at 300 rpm; thenharvested by centrifugation and ruptured using EmulsiFlex-C5 (Avestin,Ottawa, Canada).

C4-VEGF was expressed in insoluble form and purified as follows:inclusion bodies were solubilized in 8 M urea, 20 mM Tris-HCl pH 8.0,150 mM NaCl, 80 mM Na₂SO₃, 10 mM Na₂S₄O₆, 10 mM DTT, incubated for 6-8hrs at 4° C. with agitation; then supplemented with 2.5 mMTris[2carboxyethyl]phosphine (Pierce), and incubated for 16-18 hrs at 4°C. Solubilized protein was refolded via a tree-step dialysis: first,10-12 hrs at 4° C. in 10 volumes of 20 mM Tris-HCl pH 8.0, 2 M urea, 0.5M arginine, 1 mM reduced glutathione, 0.4 mM oxidized glutathione;second, 24 hrs at 4° C. in 50 volumes of 20 mM Tris-HCl pH 8.0; andthird, 24 hrs at 4° C. in 50 volumes of 20 mM NaOAc (pH 5.5). Afterdialysis, solution was clarified by centrifugation (15,000×g for 20min), and C4-VEGF was purified by chromatography on Heparin HP Sepharose(1-ml pre-packed columns, Amersham). For conjugation, C4 thiol group inC4-VEGF was deprotected by mild DTT treatment using 1.2 molar excess DTTat 34° C. for 45 min.

To minimize potential involvement of cysteine residue present inposition 116 in native VEGF 121, the inventors prepared a version ofC4-VEGF truncated at position 112 using methods known in art. Truncatedprotein contains 110 amino acids (a 3-112aa fragment of VEGF121), andtherefore it was named C4-VEGF₁₁₀. The C4-VEGF₁₁₀ was expressed andpurified as described above and displayed activities similar to that ofC4-VEGF in tissue culture assays.

To further optimize C4-tagged VEGF the inventors constructed a singlechain VEGF fused to a single C4 peptide tag via a G₄S linker peptide(C4-scVEGF). In general, engineering, expression, refolding, andpurification of monomeric proteins is simplier than oligomeric, or evendimeric, such as native isoforms of VEGF. In addition, scVEGF wasdesigned to avoid formation of inactive monomers of VEGF that areusually present in preparations of dimeric VEGF, to simplify any proteinre-engineering of VEGF, to simplify expression, refolding andpurification and to simplify conjugation of binding moieties to a singleC4-tag. First, the pET/Hu-C4-G₄S vector DNA was cut with BamHI anddephosphorylated. DNA encoding a 3-112-aa long fragment of human VEGFwas amplified from the pLen-121 plasmid using a sense primer (SEQ IDNO:33) introducing BamHI site immediately upstream of VEGF codon 3, andan antisense primer (SEQ ID NO:34) introducing a stop codon immediatelydownstream of codon 112 of VEGF and BamHI site immediately after thestop codon. The amplified DNA fragment was purified, cut with BamHI, andcloned into BamHI site of the pET/Hu-C4-G4S vector. Correct orientationof cloned ORF was selected by PCR using a T7-promoter based senseprimer. Plasmid DNA purified from the selected clones was confirmed bysequencing and designated pET/C4(G4S)VEGF₁₁₀.

SEQ ID NO:33 5′-CACGGATCCGGTGGCGGCGGTAGTGGT SEQ ID NO:345′-CACGGATCCTCATCTTGCTCTATCTTTCTTTGGTCTGC

The pET/C4(G4S)VEGF₁₁₀ vector DNA was cut with NcoI anddephosphorylated. DNA encoding a 3-112-aa fragment of human VEGF wasamplified from the pLen-121 plasmid by PCR using a sense primer (SEQ IDNO:35) introducing NcoI site immediately upstream of VEGF codon 3, andantisense primer (SEQ ID NO:36) introducing a cytosine immediatelydownstream of VEGF codon 112 (for compensation of ORF shift) and NcoIsite immediately after the introduced cytosine. The amplified DNAfragment was purified, cut with NcoI, and cloned into NcoI site of thepET/C4(G4S)VEGF₁₁₀ vector. Clones with two VEGF₁₁₀ copies were selectedby PCR using a T7-promoter based sense primer and a T7-terminator basedantisense primer. DNA isolated from four random selected clones wassequenced, and a clone containing a VEGF₁₁₀ tandem with a head-to-tailVEGF₁₁₀ orientation was selected for propagation. The selected plasmiddesignated pET/C4(G₄S)scVEGF was transformed into competent E. colicells strain BL21 (DE3) for protein expression.

SEQ ID NO:35 5′-CACAAGCCATGGCACCCATGGCAGAAGGAGGA SEQ ID NO:365′-ACTACCCATGGCTCTTGCTCTATCTTTCTTTGGTCTGC

The C4-scVEGF was expressed and purified as described above anddisplayed activities similar to that of C4-VEGF in tissue cultureassays. C4-scVEGF was recovered with ˜50% of C4 thiol group availablefor conjugation. For conjugation, C4 thiol group in C4-VEGF wasdeprotected by mild DTT treatment with 0.5 molar equivalent of DTT.

C4-LFn was expressed in a soluble cytoplasmic form and purified asfollows: soluble part of bacterial lysate was dialyzed against 200volumes of 20 mM Tris-HCl pH 8.0 for 20 hrs at 4° C., clarified bycentrifugation (15,000 g for 20 min); and passed through Sepharose Q FFcolumn. C4-LFn containing fractions were pooled together, dialyzedagainst 100 volumes of 20 mM NaOAc pH 6.5 for 20 hrs at 4° C., andpurified on Heparin HP Sepharose followed by hydrophobic interactionchromatography on Butyl FF Sepharose (1-ml pre-packed columns,Amersham). The yield of C4-LFn was 8-10 mg/L, purity >98% by SDS-PAGEfollowed by SimplyBlue Safe Stain (Invitrogen).

C4-annexin was purified as described for Hu-annexin (Backer et al.,2004). Depending on the nature of a linker, and a protein, a cysteineresidue in C4-tag is involved to a various degree in mixed disulfidewith red-ox components of the refolding buffer. When necessary, cysteineresidues can be deprotected with DTT under conditions that are optimizedfor every C4-protein.

6. Construction and Expression of Adapter Proteins.

6.1. Chimeric 1-29B/30-127H-RNase (BH-RNase) comprising of a 1-29 aafragment of bovine RNase A and a 30-127 aa fragment of human RNase I hasbeen constructed, expressed and purified as described (Gaynutdinov etal., 2003). The V118C mutation was introduced in thepET29/1-29B/30-127H-RNase plasmid DNA by site-directed mutagenesis usingGene-Tailor Site Directed mutagenesis kit (Invitrogen) and primers (SEQID NO:37 and SEQ ID NO:38). The VC118 substitution was confirmed bysequencing, the protein was designated BH-RNase(C118). BH-RNase(C118)was expressed and purified as described for wild type BH-RNase(Gaynutdinov et al, 2003). The presence of a reactive thiol group wastested by reaction with a thiol reagent N-(1-pyrene)-maleimide(Molecular Probes, Eugene, OR) followed by RP HPLC analysis as described(Backer et al., 2002). HuS(C118) adapter protein was obtained by limiteddigestion of BH-RNase(C118)with subtilisin (Sigma) as described forBH-RNase (Gaynutdinov et al., 2003).

SEQ ID NO:37 5′-AAGGCTCTCCGTACGTTCCGTGTCATTTCGACGCG SEQ ID NO:385′-CGGAACGTACGGAGAGCCTTCGCATGCAAC

6.2. HuS(C88, C118)

The N88C mutation was introduced in the pET29/1-29B/30-127H-RNase(V118C)plasmid DNA by site-directed mutagenesis using primers (SEQ ID NO:39 andSEQ ID NO:40). The N88C substitution was confirmed by sequencing, andthe protein was designated BH-RNase(C88, C118). BH-RNase(C88, C118) wasexpressed, purified and digested by subtilisin to yield HuS(C88, C118)adapter protein as described above for BH-RNase(C118).

5′-TCACTGACTGCCGTCTTACTTGCGGATCCCGTT SEQ ID NO:39 85′-AGTAAGACGGCAGTCAGTGATATGCATAGAA SEQ ID NO:40

6.3 HuR(A8, P11, C118)

The Q11P and F8A mutations were introduced consequently in thepET29/1-29B/30-127H-RNase(V118C) plasmid DNA by site-directedmutagenesis using primers SEQ ID NOS:41-44, respectively. Bothsubstitutions were confirmed by sequencing, and the protein wasdesignated BH-RNase(A8, P11,C118). BH-RNase(A8, P11, C118) was expressedand purified as described above for BH-RNase(C118).

5′-GCAGCCAAGTTTGAGCGGCCGCACATGGACTC SEQ ID NO:415′-GCCGCTCAAACTTGGCTGCTGCAGTTTCCTT SEQ ID NO:425′-GGAAACTGCAGCAGCCAAGGCTGAGCGGCCGC SEQ ID NO:435′-CTTGGCTGCTGCAGTTTCCTTCATATGTATAT SEQ ID NO:44

7. Construction and Expression of Proteins Used in the Assays.

7.1. SLT-VEGF.

The pET/VEGF121-SLT/L plasmid encoding SLT-VEGF fusion protein carryingN-terminal S-tag and His-tag was described previously (Backer & Backer,2001). The SLT-VEGF DNA coding sequence was amplified by PCR from thepET/VEGF121-SLT/L plasmid DNA using primers (SEQ ID NO:45) introducingNdeI site and (SEQ ID NO:46) introducing Xho I site. Purified PCRproduct was cut with Nde I and Xho I restrictases, purified, and clonedinto Nde I-Xho I sites of the pET29a(+) vector (Novagen). The resultingplasmid was confirmed by sequencing and designated pET29/SLT-VEGF.SLT-VEGF was expressed in BL21(DE3) as described above for C4-carryingproteins, and purified as follows: inclusion bodies were solubilized in8 M urea, 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 80 mM Na2SO3, 10 mMNa₂S₄O₆, 10 mM DTT and incubated for 6-8 hrs at 4° C. with agitation;then supplemented with 5 mM Tris[2carboxyethil]phosphine (Pierce) andincubated for 16-18 hrs at 4° C. Solubilized protein was refolded via atree-step dialysis: first, for 8-10 hrs at 4° C. in 10 volumes of 20 mMTris-HCl pH 8.0, 2 M urea, 0.5 M arginine, 1 mM reduced glutathione, 0.4mM oxidized glutathione; second, 24 hrs at 4° C. in 100 volumes of 20 mMTris-HCl pH 8.0, 150 mM NaCl, 0.01% NP-40; and third, 24 hrs at 4° C. in100 volumes of 20 mM Tris-HCl pH 8.0, 20 mM NaCl. After dialysis,solution was clarified by centrifugation (15,000×g for 20 min), andSLT-VEGF was purified by chromatography on HiTrap Q Sepharose (1-mlpre-packed columns, Amersham).

SEQ ID NO:45 5′- AAAAACATATGAAGGAATTTACCTTAGACTTCTCG SEQ ID NO:46 5′-TACTCGAGTCACCGCCTCGGCTTGTCAC

7.2. Hu-VEGF.

Hu-VEGF 121 fusion protein carrying N-terminal wild type Hu-tag wasconstructed as described in Backer et al., 2003. Hu-VEGF121 wasexpressed and refolded from inclusion bodies as described above forC4-VEGF with the following modifications: refolding from inclusionbodies was done via a two-step dialysis: first, for 8-10 hrs at 4° C. in10 volumes of 20 mM Tris-HCl pH 8.0, 2 M urea, 0.5 M arginine, 1 mMreduced glutathione, 0.4 mM oxidized glutathione; second, 24 hrs at 4°C. in 50 volumes of 20 mM Tris-HCI pH 8.0. Also, for final Hu-VEGF121purification, HiTrap SP Sepharose FF followed by Heparin HP Sepharosechromatography (1-ml pre-packed columns, Amersham) was performed.

8. Functional Activities of Recombinant Fusion Proteins with C4-Tag.

A. VEGF activity. Functional activities of VEGF and VEGF-basedconjugates were tested in two assays. First assay, stimulation ofVEGFR-2 autophosphorylation was performed as follows: near-confluent293/KDR cells after overnight starvation (DMEM/0.5% FBS) were shifted toserum-free DMEM with 0.5 mM sodium vanadate for 20 min at 37° C., thenstimulated with VEGF for 10 min at 37° C., lysed and analyzed by Westernblotting using anti-phosphotyrosine RC20:HRPO conjugate (BD TransductionLabs, USA). Second assay, protection of 293/KDR cells from cytotoxiceffect of SLT-VEGF, was performed as follows. 293/KDR cells were platedon 96-well plates 20 hrs before the experiment, 1000 cells/well. Varyingamounts of VEGF or VEGF-based conjugates were mixed with SLT-VEGF incomplete culture medium, and added to cells in triplicate wells to afinal SLT-VEGF concentration of 1 nM. Viable cells were quantitated 96hrs later by CellTiter 96® AQueous One Solution Cell Proliferation Assaykit (Promega, USA).

B. Annexin V activity. Functional activities of C4-annexin andHuS-C4-annexin were tested by their ability to compete with FITC-annexin(Sigma) for binding to phosphatidylserine-displaying erythrocytes ofstabilized human blood (4 C Plus Cell Control, Beckman Coulter, USA) asdescribed (Tait et al., 1995). Briefly, ten million erythrocytes wereincubated with varying amounts of annexin in the presence of 5 nMFITC-annexin V (Sigma, St. Louis, Md.) in binding buffer (10 mM HEPES,pH 7.4, 136 mM NaCl, 2.5 mM CaCl2) for 15 min at RT. Cells were spundown, resuspended in a binding buffer supplemented with 5 mM EDTA, andspun down again. FITC-annexin fluorescence in the supernatants wasmeasured at λ_(ex) 485 nm/λ_(em) 520 nm.

C. LFn activity. C4-LFn and Hus-C4-LFn were tested for their ability toprotect RAW cells from cytotoxic effects of full-length LF in thepresence of PA. RAW264.7 cells were plated on 96-well plates 15×10³cells/well 20 hrs before the experiment. Varying amounts of LFn, C4-LFn,or Hus-C4-LFn were mixed with LF and PA (List Biological, USA) in DMEMcomplete culture medium, and added to cells in triplicate wells to finalconcentrations of 2 nM PA and 0.2 nM LF. After 2.5 hrs of incubation at37° C. in 5% CO₂, viable cell numbers were determined by CellTiter 96®AQueous One Solution Cell Proliferation Assay kit (Promega).

9. Cell Lines.

HEK293 human transformed embryonic kidney cells (CRL-1573) and RAW 264.7mouse monocytes (TIB-71) were from American Type Culture Collection(ATCC, Rockville, Md.). 293/KDR cells expressing 2.5×10⁶ VEGFR-2/cellhave been developed in SibTech, Inc. (Newington, Conn.; Backer andBacker, 2001a). All cells were maintained in DMEM (Life Technologies,USA) supplemented with 10% fetal calf serum (Gemini, USA), 2 mMglutamine (Life Technologies, USA), and penicillin-streptomycin (LifeTechnologies, USA) at 37° C., 5% CO2.

Example 2 Site-Specific Conjugation of C4-Tagged Proteins to AdapterProtein HuS(C118)

The protocol included site-specific conjugation of C4-tagged proteins toadapter protein HuS(C118) and testing the functional activities ofconjugates in tissue culture.

HuS(C118) prepared as described above, and C4-tagged proteins, such asC4-VEGF, or C4-annexin, or C4-LFn, were mixed in a buffer containing 20mM Tris HCI pH 8.0, incubated at 4° C. for 16 hrs, and then analyzed bySDS-PAGE under reducing and non-reducing conditions. SDS-PAGE analysisunder non-reducing conditions revealed new protein products formed inthe mixtures during incubation (FIG. 2C, no DTT lanes). The molecularweights of the proteins in these bands corresponded to the molecularweight of conjugates carrying one or two HuS molecules per parentalprotein, respectively. Under reducing conditions these bandsdisappeared, and only bands corresponding to parental proteins and12-kDa HuS(C118) were detectable (FIG. 2C, plus DTT lanes). Conjugateswere purified first on Hu-peptide column to remove free HUS(C118) and,when necessary, by ion-exchange chromatography to remove free parentalproteins.

The functional activities of conjugates were tested in tissue cultureassays described above. Activity of HuS-C4-VEGF conjugate was similar tothat of C4-VEGF in the induction of VEGFR-2 tyrosine autophosphorylationin 293/KDR cells (FIG. 3, Panel A) HuS-C4-annexin conjugate was activein competition with FITC-annexin for binding tophosphatidylserine-displaying erythrocytes of stabilized human bloodwith IC50 of 11+3 nM for conjugate vs. IC50 of 9+4 nM for recombinantannexin V as reported by Tait et al., (1995) (FIG. 3, Panel B).HuS-C4-LFn was as active as C4-LFn in protection of RAW 264.7 cells fromLF in the presence of PA (FIG. 3, Panel C). Together, these dataindicate that conjugation of adapter protein to different C4-taggedprotein does not destroy their activity.

Example 3 Site-Specific Conjugation of C4-VEGF and C4-scVEGF toRadionuclide Chelator, 5-Maleimido-2-Hydraziniumpyridine Hydrochloride

The protocol included site-specific conjugation of C4-VEGF and C4-scVEGFto chemically active radionuclide chelator5-maleimido-2-hydraziniumpyridine hydrochloride (Solulink, San Diego,Calif.), and testing the conjugate in tissue culture.

C4-VEGF, prepared as described above, was mixed with dimethylformamidedissolved 5-maleimido-2-hydraziniumpyridine hydrochloride hereinafterdesignated as HYNIC at the molar ratio HYNIC/protein 3:1 in a buffercontaining 20 mM Tris HCl pH 8.0 and incubated for one hour at roomtemperature. The product, designated HYNIC-C4-VEGF, was purified onPD-10 column (Amersham, USA) equilibrated with 0.114 M Tricine, pH 6.9.Analytical RP HPLC (FIG. 4, Panel A) was used to determine theconcentration of protein by detection of optical density at 216 nm andthe concentration of HYNIC by detection of optical density at 310 nm.Under selected conditions the ratio of HYNIC to C4-VEGF in purifiedHYNIC-C4-VEGF was ˜1.

C4-scVEGF, prepared as described above, was conjugated to HYNIC andpurified as described above and the ratio of HYNIC to C4-scVEGF inpurified product designated HYNIC-C4-VEGF, was ˜1.

The functional activity of VEGF moiety in HYNIC-C4-VEGF andHYNIC-C4-scVEGF was tested in two tissue culture assays described above,induction of VEGFR-2 tyrosine autophosphorylation in 293/KDR cells(FIG.4, Panel B) and protection of 293/KDR cells from cytotoxic activity ofSLT-VEGF (FIG. 4, Panel C). In both assays activities of HYNIC-C4-VEGFand HYNIC-C4-scVEGF were comparable to that of unmodified C4-VEGF,indicating that conjugation of C4-VEGF or C4-scVEGF to HYNIC does notdestroy activity of the protein.

Example 4 Site-Specific Conjugation of C4-VEGF to Polyethyleneglycol

The protocol included site-specific conjugation of C4-VEGF₁₁₀ topolyethyleneglycol functionalized with chemically active maleimide group(Nektar Therapeutics) and testing the conjugate in tissue culture.

C4-VEGF₁₁₀, prepared as described above, was mixed with either 20 kDa or40 kDa polyethyleneglycol maleimide (PEG) at PEG to protein ratio of 3:1and incubated for one hour at room temperature in a buffer containing 20mM Tris HCl pH 8.0. The products, designated PEG20-C4-VEGF andPEG40-C4-VEGF, were purified from unreacted C4-VEGF and PEG by HPLCgel-filtration on a column equilibrated with 20 mM Tris HCI pH 8.0.Analytical RP HPLC was used to determine the concentration of protein bydetection of optical density at 216 nm. Western blot analysis of reducedPEGylated VEGF products with antibody against VEGF revealed a bandcorresponding to apparent molecular mass of 55 kDa and a band ofapproximately equal intensity corresponding to unmodified VEGF monomer(FIG. 5, Panel A), indicating that in the majority of the VEGF dimersonly one C4-tag was conjugated to PEG.

The functional activity of PEG-C4-VEGF was tested in tissue cultureassay of induction of VEGFR-2 tyrosine autophosphorylation in 293/KDRcells (FIG. 5, Panel B for PEG40-C4-VEGF) In this assay activities ofPEG-C4-VEGF were comparable to that of unmodified C4-VEGF, indicatingthat site-specific conjugation of C4-VEGF to polyethyleneglycol does notdestroy activity of the protein.

Example 5 Site-Specific Conjugation of Cyanine Dye Cy5.5 To HuS-C4-VEGFConjugate

The protocol included preparation of HuS(C88, C118), its conjugation toC4-VEGF, purification of the resulting Hus-C4-VEGF conjugate, andconjugation of said conjugate to a cyanine dye Cy5.5 yielding conjugatenamed Cy5.5-Hus-C4-VEGF (FIG. 6, Panel A). For comparative purposes,C4-VEGF was either randomly modified with NHS-Cy5.5 on amino groups tothe ratio of 1:1, or modified with maleimide-Cy5.5 on C4 residue in theC4-tag. The functional activities of VEGF moiety in all Cy5.5-containingconjugates were tested in two tissue culture assays described above,induction of VEGFR-2 tyrosine autophosphorylation (FIG. 6, Panel B) andprotection of 293/KDR cells from cytotoxic activity of SLT-VEGF (FIG. 6,Panel C). In both assays only activities of Cy5.5-Hus-C4-VEGF conjugatewere comparable to that of unmodified C4-VEGF, underlying efficacy ofusing an adapter protein capable of binding to C4-tag for derivatizationof proteins.

The utility of Cy5.5-Hus-C4-VEGF for imaging was tested in Balb/c femalemice bearing subcutaneous 4T1 mouse mammary adenocarcinoma tumors.Images obtained with KODAK Image Station 2000 MM equipped with aband-pass filter at 630 nm and a long-pass filter at 700 nm indicatedthat Cy5.5-HuS-C4-VEGF conjugate preferentially localized to theperiphery of primary tumor (FIG. 6, Panel D). This preferentiallocalization was inhibited in mice pretreated with SLT-VEGF protein thatdestroys VEGFR-2 positive cells in tumors (Backer et al., submitted),indicating that accumulation of Cy5.5-HuS-C4-VEGF conjugate in tumor isVEGF-receptor mediated and can be used for specific imaging of tumorvasculature.

Example 6 The Use of C4-Tag and an Adapter Protein Covalently Bound tothis Tag for Construction of VEGF-Driven Conjugates ContainingDrug-Loaded Liposomes for Targeted Drug Delivery

The protocol included preparation of HuS(C118), its conjugation toPEG-lipid-maleimide, insertion of lipidated HuS(C118) intodoxorubicin-loaded liposomes, that leads to a standardized constructnamed Lip/HuS(C118), and conjugation of this construct to C4-VEGF, thatyielded Lip/Hus-C4-VEGF (FIG. 7, Panel A). The functional activity ofVEGF moiety in Lip/Hus-C4-VEGF conjugate were tested in a tissue cultureassay described above, induction of VEGFR-2 tyrosine phosphorylation in293/KDR cells (FIG. 7, Panel B). In this assay VEGF activity ofLip/Hus-C4-VEGF liposomes was comparable to that of unmodified C4-VEGF,underlying efficacy of using an adapter protein capable of binding toC4-tag for derivatization of proteins.

A 15-min exposure of 293/KDR cells to Lip/HuS-C4-VEGF resulted in adose-dependent inhibition of cell growth (FIG. 7, Panel C), whileequivalent amounts of untargeted doxorubicin-loaded liposomes(commercially available under the trade name “DOXIL”) derivatized withHuS(C118) alone were not toxic for these cells indicating a VEGFreceptor-mediated mechanism of cytotoxicity of Lip/Hus-C4-VEGF targetedliposomes. This mechanism was further confirmed by ability of VEGF toinhibit of cytotoxicity of Lip/Hus-C4-VEGF in a dose-dependent manner(FIG. 7D).

Example 7 The Use of C4-Tag for Construction of Scvegf-Driven ConjugatesContaining Drug-Loaded Liposomes for Targeted Drug Delivery.

The protocol included preparation of C4-scVEGF its conjugation toPEG-lipid-maleimide, and insertion of lipidated C4-scVEGF intodoxorubicin-loaded liposomes (“DOXIL”) (FIG. 8, Panel A). The functionalactivity of VEGF moiety in Lip/C4-scVEGF conjugate were tested in atissue culture assay described above, induction of VEGFR-2 tyrosinephosphorylation in 293/KDR cells (FIG. 8, Panel B). In this assay VEGFactivity of Lip/C4-scVEGF liposomes was comparable to that of unmodifiedC4-VEGF, underlying efficacy of using C4-scVEGF.

A 15-min exposure of 293/KDR cells to Lip/C4-scVEGF resulted in adose-dependent inhibition of cell growth (FIG. 8, Panel C), whileequivalent amounts of untargeted doxorubicin-loaded liposomes (“DOXIL”)were not toxic for these cells indicating a VEGF receptor-mediatedmechanism of cytotoxicity of Lip/C4-scVEGF targeted liposomes. Thismechanism was further confirmed by ability of VEGF to inhibit ofcytotoxicity of Lip/Hus-C4-VEGF in a dose-dependent manner (FIG. 8D).

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1. A biological conjugate, comprising: (a) a targeting moiety comprisinga polypeptide having an amino acid sequence comprising SEQ ID NO:2 andthe polypeptide sequence of a selected targeting protein selected fromthe group consisting of human vascular endothelial growth factor (VEGF)comprising the sequence of SEQ ID NO: 6 or SEQ ID NO: 8, human annexin Vcomprising the sequence of SEQ ID NO: 10, a catalytically inactivefragment of anthrax lethal factor comprising the sequence of SEQ ID NO:12, and a single chain vascular endothelial growth factor (scVEGF)comprising the protein sequence of SEQ ID NO: 4 or SEQ ID NO: 14; and(b) a binding moiety selected from the group consisting of drugs,radionuclide chelators, polyethylene glycol, dyes, lipids, liposomes, asurface of a nano- or microparticle, a surface of a dendrimer, a surfaceof a tissue scaffold, a surface of a biomedical device, and a surface ofa biosensor; said biological conjugate having a covalent bond betweenthe thiol group of SEQ ID NO:2 and said binding moiety.
 2. Thebiological conjugate of claim 1, wherein said single chain vascularendothelial growth factor (scVEGF) comprises the sequence of SEQ IDNO:14.
 3. A biological conjugate, comprising: (a) a targeting moietycomprising a polypeptide having an amino acid sequence comprising SEQ IDNO:2 and the polypeptide sequence of a selected targeting proteinselected from the group consisting of human vascular endothelial growthfactor (VEGF) comprising the sequence of SEQ ID NO: 6 or SEQ ID NO: 8,human annexin V comprising the sequence of SEQ ID NO: 10, acatalytically inactive fragment of anthrax lethal factor comprising thesequence of SEQ ID NO: 12, and a single chain vascular endothelialgrowth factor (scVEGF) comprising the protein sequence of SEQ ID NO: 4or SEQ ID NO: 14; and (b) a binding moiety comprising a complimentaryadapter protein covalently bound to said targeting moiety, and whereinsaid adapter protein is a fragment of mutant human Rnase I comprisingCys₁₁₈; said biological conjugate having a covalent bond between thethiol group of SEQ ID NO:2 and said binding moiety.
 4. The biologicalconjugate of claim 3, wherein said adapter protein has a polypeptidesequence selected from the group consisting of SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, and SEQ ID NO:22.
 5. A pharmaceutical compositioncomprising: (a) a pharmaceutically acceptable carrier; and' (b) thebiological conjugate of claim 1.